The L. intracellularis strain (a commercial live attenuated L. intracellularis vaccine, Enterisol®ileitis) was purchased from Boehringer Ingelheim Vetmedica, Germany, and Escherichia coli (CAU0751), Salmonella choleraesuis (CVCC2139), and Salmonella typhimurium (CVCC2220) were purchased from the China Veterinary Culture Collection Center (CVCC). Desulfovibrio (ATCC 27774) and Bilophila wadsworthia (ATCC 49260) were purchased from the American Type Culture Collection (ATCC). Brachyspira hyodysenteriae is courtesy of the Shanghai Animal Disease Prevention and Control Center. The pGex-6p-1 (+) vector was purchased from Novagen, USA. The myeloma cell line SP2/0 (stored in our laboratory) and rat small intestine cells (IEC-18; ATCC CRL 1589) were cultured in DMEM/high glucose (Gibco, USA) in a humidified 5% CO₂ atmosphere at 37°C. All the cell culture media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA). L. intracellularis was grown as described (5). In brief, 30% confluent IEC-18 monolayers were infected with L. intracellularis and incubated in an atmosphere of 83.2% nitrogen (N₂), 8.8% carbon dioxide (CO₂), and 8% oxygen (O₂) for 7 days at 37°C.

The outer membrane protein (Omp2) gene sequences of L. intracellularis were selected and analyzed. The partial Omp2 sequence GXNN strain was synthesized according to the 103–993 bp regions from L. intracellularis (no. EU621796.1) by Sangon Biotech Co., Ltd. (Shanghai, China) using restriction enzymes (BamH I and Sal I). The synthetic Omp2 gene was cloned into the prokaryotic expression vector pGex-6p-1 (+). The positive recombinant plasmids were identified by enzyme digestion and sequencing and then transformed into E. coli Rosetta (DE3) competent cells. The expression of the recombinant Omp2 protein was induced with 0.3 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, USA) at 16°C for 18 h, and the protein was examined by SDS-PAGE. The recombinant protein was also detected by SDS-PAGE after purification with a high-affinity GSTRAP HP column (GE Healthcare Life Sciences, USA) according to the instructions of the manufacturer, and the concentration was determined with a NANODROP 2000 Spectrophotometer (Thermo, USA). In addition, Western blotting was performed to evaluate the reactivity of the purified Omp2 protein. The same method was described in the reference with some modifications (25). Briefly, the purified Omp2 protein was transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, USA) after being separated by 12% SDS-PAGE. Then the membrane was blocked with 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.01% Tween-20, pH 7.4) overnight at 4°C, followed by incubation with an anti-L. intracellularis-positive antibody (SVANOVIR® L. intracellularis/Ileitis-Ab, Germany) for 1 h at 37°C. After three washes with TBST, the membrane was incubated with goat anti-pig IgG H&L (HRP) secondary antibody for 1 h at 37°C (Zhuangmeng, China). After three washes with TBST, the target protein bands were detected using 3,3-diaminobenzidine tetrahydrochloride (DAB), and the reaction was stopped by rinsing with distilled water and drying the membrane.

The animal protocol used was approved by the Ethical Committee of the Faculty of Veterinary Science of Nanjing Agricultural University (approval number: IACUC20181009); and all experiments were performed in accordance with the relevant guidelines and regulations. The purified Omp2 protein was used to immunize 6-week-old BALB/c mice (Yangzhou University Comparative Medical Center, China). The mice were immunized with Freund's adjuvant (Sigma, USA) three times. Three days after the final immunization, the mice with the highest antibody titers were euthanized, and the spleen cells were harvested. Subsequently, splenocytes from immunized mice were fused with sp2/0 murine myeloma cells by using 50% polyethylene glycol (PEG 4000, Sigma, USA) according to a previously described method (26). The fusion cells were separated into 96-well-plates and cultured selectively in DMEM/high glucose containing 10% fetal bovine serum, 100 mM hypoxanthine, 400 mM aminopterin, and 16 mM thymidine. On day 5, 50 μl of HAT medium was added to each well of the 96-cell plates. On day 12, the HAT medium was completely replaced with fresh HT medium. After HAT/HT medium screening, the culture supernatants were analyzed by indirect enzyme-linked immunosorbent assay (ELISA). The positive hybridoma cells were repeatedly subcloned by the limiting dilution method until monoclonal hybridoma cells were obtained. Positive hybridoma cells were cultured in the abdominal cavity of paraffin-primed BALB/c mice to obtain ascitic fluid. The characteristics of the monoclonal antibodies (mAbs) were identified using a Pierce Rapid Isotyping Kit (Thermo Scientific, USA) according to the instructions of the manufacturer.

The antibody titers of ascitic fluid were further determined by ELISA, as previously described (27). Briefly, the purified recombinant Omp2 protein was used as a coating antigen, the ascitic fluids were diluted in serial 2-fold dilutions from 1:1,000 to 1:2,048,000 and added to the wells in triplicate, and the negative control (unimmunized normal mouse serum) was included in each plate. After washing five times with PBST (0.5% Tween-20 in PBS), the plate was incubated with HRP-conjugated goat anti-mouse IgG (Abmart, China) at a ratio of 1:5,000 for 1 h at 37°C. The color was developed with 3,3′,5,5′- tetramethylbenzidine (TMB), and the reaction was stopped by 2 M H₂SO₄. The OD_450nm was measured in a micro titer plate reader (Bio-Rad Benchmark Plus). The cutoff value was determined when the OD_450nm ratio (OD_450nm of purified antibody/OD_450nm of unimmunized rabbit serum) was above 2.1. The antibody titer was defined as the highest dilution that yielded a net OD_450nm value greater than the calculated cutoff.

The expressed Omp2 proteins were subjected to gel electrophoresis on 12% SDS-PAGE after denaturation with 2 × SDS loading buffer at 100°C for 3~5 min. The protein bands were transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST overnight at 4°C. After washing three times in TBST, the membranes were incubated with mAbs against Omp2 protein, positive control (mouse serum against Omp2 protein, collected from the last injection) or negative control (unimmunized normal mouse serum) at 37°C for 1 h. After washing with TBST, the membranes were immersed in goat anti-mouse HRP-conjugated polyclonal serum at 37°C for 1 h. After washing in the same way, the membranes were immersed in DAB, and the reaction was stopped by rinsing with distilled water and drying the membrane.

The cross-reactivity of the mAbs was detected by IFA. Briefly, about 1 × 10⁵ L. intracellularis, 1 × 10⁵ CFU of Desulfovibrio, B. wadsworthia, S. choleraesuis, S. typhimurium, E. coli, and B. hyodysenteriae were tested using a bacterial suspension to infect IEC-18 cells. The cell cultures were detected after being incubated at 37°C in 8.0% O₂, 8.8% CO₂, and 83.2% N₂ in a humidified incubator for 3 h, the rest of the experiment steps were the same as before.

Immunofluorescence assays were performed using IEC-18 cells seeded on 13-mm sterilized coverslips and cultured overnight to 30% confluence. The cells were infected with 1 × 10⁵ L. intracellularis at a multiplicity of infection (MOI) of 100 for 3 h and 7 days in a tri-gas incubator with 83.2% N₂, 8.8% CO₂, and 8% O₂ at 37°C (Gene Science AG300 plus, USA) according to methods described in the literature (28). Uninfected cells were used as negative control. The cells were washed with PBS three times and fixed with ice-cold 4% paraformaldehyde (−20°C) for 15 min, and the cells were permeabilized with 0.3% TritonX-100 for 10 min at room temperature. After blocking for 2 h at 37°C in 5% skim milk in PBS, the fixed cells were then incubated with a 1:200 dilution of mAbs for 1 h at 37°C. Unimmunized normal mouse serum and mouse serum against the Omp2 protein were used as negative and positive controls, respectively. The cell monolayers were rehydrated by rinsing three times with PBST. FITC-labeled goat anti-mouse IgG and goat anti-mouse IgG Alexa Fluor 594 were used as a secondary antibody, respectively, at a 1:1,000 dilution and incubated in the dark for 1 h at 37°C. After three washes with PBST, 200 μl of DAPI (4,6-diamidino-2-phenylindole dihydrochloride; Ziyi Reagent Co., Shanghai, China) staining solution was added to each well, and the nuclei were stained in the dark for 10 min at room temperature. After washing, the coverslips were removed and immediately examined by confocal laser scanning microscope (Nikon ECLIPSE Ti, Japan).

The monoclonal antibody was also evaluated by immunofluorescence (IF) staining and immunocytochemistry (IHC) in histological fragments of lesions suggestive of PPE. Briefly, infected (subclinical form of PE) and normal intestinal samples were obtained from commercial pigs in a slaughterhouse in Jiangsu Province, China. Subsequently, the samples of ileum were fixed by immersion in 10% neutral-buffered formalin. L. intracellularis-specific Omp2 monoclonal antibody (4D9, 3G2, and 7G5) was used to perform IF and IHC to stain L. intracellularis bacteria according to the previously reported methods (29, 30). Uninfected sample was used as negative controls at the same time.

The Omp2 gene was correctly inserted into the pGex-6p-1 vector according to nucleotide sequencing and restriction analysis (Figure 1, lane 2). The sequence analysis of the protein was performed by DNAStar software. MegAlign analysis results showed that the homology of the amino acid sequence between GXNN strain outer membrane protein and LI0902 of PHE/MN1-00 (GenBank No. AM180252.1) is 99.1%. Compared with LI0902 of PHE/MN1-00, the 175th (Val to Alu), 223th (Glu to Gly), and 254th (Leu to Ser) amino acids generated mutation amino acids from the GXNN strain. Subsequently, the recombinant Omp2 protein was successfully expressed in BL21 (DE3) cells after it was induced by IPTG. The expressed recombinant GST-Omp2 protein was ~58.6 kDa and present in the supernatant of bacterial lysates (Figure 2A, lane 1). After purification by a GSTRAP HP column, the purity of the final products in the portion of the eluate was ~98%, as determined by image analysis of Coomassie blue-stained SDS-PAGE (Figure 2A, lane 2). Western blotting assays showed that the purified Omp2 could be specifically recognized by the anti-L. intracellularis-positive antibody (Figure 2B, lane 1), whereas no reactive band appeared when purified GST was used as the negative control (Figure 2B, lane 2).

Purified Omp2 proteins were used to immunize BALB/c mice. To avoid obtaining mAbs against the GST tag, the GST tag of Omp2 was digested using PreScission Protease (Beyotime Biotechnology. Inc., China) before it was used as a coating antigen in the ELISA for screening (data not shown). Finally, three hybridoma cell lines named 4D9, 3G2, and 7G5 were acquired by subcloning and screening. The titers of ascitic fluids were measured by indirect ELISA, and the titers of 4D9, 3G2, and 7G5 were 1:2,048,000, 1:512,000 and 1:256,000, respectively (Figure 3).

The results of subtype identification showed that the heavy chains of the three mAbs (4D9, 3G2, and 7G5) all belonged to IgG1, and the light chains were of the kappa type (Table 1).

Western blotting analysis showed that all the mAbs and the positive control (the mouse serum against Omp2 protein) could react with the Omp2 protein (Figure 4, lane 1–4), whereas no reactive band appeared when the negative control (unimmunized normal mouse serum) was used (Figure 4, lane 5).

To test the specificity of the 4D9, 3G2, and 7G5, the IFA was conducted with pure cultures of the following bacterial strains: Desulfovibrio, B. wadsworthia, S. choleraesuis, S. typhimurium, E. coli, and B. hyodysenteriae. The results showed that 4D9, 3G2, and 7G5 did not have cross-reactions with enteric bacteria commonly found in the ileum of pigs or closely related to L. intracellularis (Figure 5). The results indicated that the new monoclonal antibody prepared in this study is specific against L. intracellularis.

The mAbs could react with L. intracellularis in the infected monolayer. In particular, significant specific signals were detected using the primary antibodies against Omp2 (4D9, 3G2, and 7G5). Our findings indicated that the L. intracellularis in the monolayer of eukaryotic cells could be detected at 3 h post infection (Figure 6A), and highly infected cells (HIC) may be observed once the bacteria are cultivated for up to 7 days post incubation. However, no signal was detected in the negative control (Figure 6B). IFA and IHC results indicated that L. intracellularis antigen was visualized mainly in the crypt of the ileum from infected tissues of PPE after it reacted with L. intracellularis-specific monoclonal antibody, while control ileum was negative (Figures 7A,B). These results demonstrated that the new monoclonal antibody could also be applied to evaluate L. intracellularis antigen in infected tissues of PPE.